Rapid C-ELISA process and related compositions

ABSTRACT

The present invention provides improved and rapid detection methods for an antigen such as a chemical compound, a peptide, a nucleic acid, or a protein released from cells or virus particles in situ. The detection time for an antigen can be dramatically reduced relative to conventional technologies. The technology can particularly be used, for example, to modify and reduce the detection time significantly in traditional ELISA, and also Western blot or Dot blot assays. The improved ELISA method is rapid, economical, reproducible, simple and automatable. Also provided are compositions and kits for using the improved ELISA methods for the rapid detection of antigens.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 12/119,793, filed May 13, 2008, which claims priority to U.S.Provisional Application November 60/929,206, filed Jun. 18, 2007, whichis incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a detection method for an antigen suchas a chemical compound, a peptide, a protein, an RNA, a DNA, a cell(proteins released in situ), or a virus particle (proteins released insitu). In particular, the present invention provides a method andcomposition useful for performing ELISA assays, which also can be usedfor Western blot and Dot blot assays.

2. Description of the Prior Art

Immunological methods have become important tools useful for detectingantigens including, for example, peptides, proteins, nucleic acids,biological cells, and virus particles. A wide variety of methods havebeen developed for the detection or quantitation of antigens. Amongthem, Western Blot, Dot Blot, ELISA and Immunohistology are the fourmost commonly used methods.

Enzyme-linked Immunosorbent Assays (ELISAs), which combine the highspecificity of antibodies with the high sensitivity of enzyme assays byusing antibodies or antigens coupled to an easily assayed enzyme thatpossesses a high turnover number such as alkaline phosphatase (AP) orhorseradish peroxidase (HRP), are very useful tools both for determiningantibody concentrations (antibody titer) in sera as well as fordetecting the presence of antigen.

There are two main variations on this method: ELISA can be used todetect the presence of antigens that are recognized by a detection agentor it can be used to test for detection agents that recognize anantigen. There are many different types of ELISAs. Four of the mostcommon types of ELISA are “Direct ELISA,” “Indirect ELISA,” “SandwichELISA” and Cell-based ELISA (C-ELISA).

A conventional direct ELISA (FIG. 1) is comprised of the followingsteps: (i) coating a solid phase with an antigen dissolved in a coatingbuffer; (ii) incubating the solid phase from Step (i) with a blockingreagent for 1 hour to block non-specific binding sites on the solidphase; (iii) washing the solid phase from Step (ii) three times with PBSor PBST for 1 min each; (iv) incubating the solid phase from Step (iii)with a primary detection agent which binds to the antigen; (v) washingthe solid support from Step (iii) five times for 1 min each in PBS orPBST to remove the non-specifically bound primary detection agent; and(vi) using a detection system such as UV, fluorescence,chemiluminescence or other detection methods to detect the bound primarydetection agent. The primary detection agent can be, without limitation,a detection agent linked (coupled) to a fluorescent dye, or a reporterenzyme such as alkaline phosphatase (AP) or horseradish peroxidase(HRP), which can convert a colorless substrate to a colored productwhose optical densities can be measured on an ELISA plate reader attarget wavelengths.

A conventional indirect ELISA (FIG. 3) is comprised of the followingsteps: (i) coating a solid phase with an antigen dissolved in a coatingbuffer; (ii) incubating the solid phase from Step (i) with a blockingreagent for 1 hour to block non-specific binding sites on the solidphase; (iii) washing the solid phase from Step (ii) three times with PBSor PBST for 1 min each; (iv) incubating the solid phase from Step (iii)with a primary detection agent diluted in a solution for 1 hour; (v)washing the solid support from Step (iv) three times for 1 min in PBS orPBST to remove the non-specifically bound primary detection agent; (vi)incubating the solid support from step (v) with a secondary detectionagent diluted in a solution for 1 hour; (vii) washing the solid supportfrom Step (vi) five times for 1 min each in PBS or PBST to remove thenon-specifically bound secondary detection agent; and (viii) using adetection system such as UV, fluorescence, chemiluminescence or othermethods to detect the bound secondary detection agent. The secondarydetection agent binds the primary detection agent. The secondarydetection agent can be, without limitation, a detection agent linked(coupled) to a reporter enzyme such as alkaline phosphatase (AP) orhorseradish peroxidase (HRP), which can convert a colorless substrate toa colored product whose optical densities can be measured on an ELISAplate reader at target wavelengths.

The complete direct ELISA procedure involves at least three incubationsteps: the first is incubation between the solid support and theantigen; the second is incubation between the solid support and theblocking reagent; and the third one is incubation between the solidsupport and the primary detection agent. The incubation step is atwo-phase reaction and involves the binding reaction between the antigenon the solid support and the detection agent.

The complete indirect ELISA procedure involves at least four incubationsteps: the first is incubation between the solid support and an antigen;the second is incubation between the solid support and the blockingreagent; the third one is incubation between the solid support and theprimary detection agent; and the fourth is incubation between the solidsupport and the secondary detection agent. The incubation step is atwo-phase reaction and involves the binding reaction between the antigenon the solid support and the detection agent.

In a conventional direct ELISA, the first incubation step, antigencoating, takes at least 2 hours and each other incubation step takesabout 1 hour. A conventional direct ELISA, as described above,therefore, takes at least 4 hours.

In a conventional indirect ELISA, the first incubation step, antigencoating, takes at least 2 hours and each other incubation step takesabout 1 hour. A conventional indirect ELISA, as described above, willtake at least 5 hours. Because conventional direct and indirect ELISAconsumes valuable time, there is a need for a simple and rapid processto address these conventional time-consuming assays.

The cell-based ELISA (C-ELISA) is a moderate throughput format fordetecting and quantifying cellular proteins including post-translationalmodifications associated with cell activation (e.g., phosphorylation anddegradation). Cells are plated, treated according to experimentalrequirements, fixed directly in the wells, and then permealized. Afterpermealizing, fixed cells are treated similar to a conventionalimmunoblot, including blocking, incubation with a first antibody,washing, incubation with a second antibody, addition of chemilumescentsubstrates and development.

In 1971, Engvall and Perlmann (Immunochem., 8:871-874, 1971) coined theterm “enzyme-linked immunosorbent assay,” which is better known by theacronym “ELISA”, to describe an enzyme-based immunoassay method which isvery useful for measuring antigen concentrations. Since then, ELISA hasnot only become one of the most commonly used methods for protein andantibody detection and identification but also the basic immunoassayupon which many of the modern assays are based.

A rapid method for microwave mediated enzyme-linked immunosorbent assay(M-ELISA) (U.S. Pat. No. 6,498,016) was developed to perform ELISArapidly. However, this procedure requires a carefully-controlledmicrowave which needs optimization.

Although there have been substantive improvements in all of theseimmuno-detection methods, including the quality of reagents, solid phaseplates and plastics, microplate readers, washers, and statisticalsoftware, the basic methodology has remained virtually unchanged.

SUMMARY OF THE INVENTION

The present invention provides improved methods and compositions forperforming a rapid enzyme-linked immunosorbent assay (ELISA).

In an aspect of the present invention, there is provided a method forperforming a direct rapid enzyme-linked immunosorbent assay (ELISA),comprising the steps of coating a solid phase with an antigen dissolvedin a quick coating buffer for between about two to twenty minutes,preferably for about five minutes; blocking the solid phase with ablocking reagent dissolved in a quick blocking buffer for between abouttwo to ten minutes, preferably for about five minutes; incubating thesolid phase with a primary detection agent in solution; washing thesolid phase to remove any unbound primary detection agent; and detectingthe presence of an agent bound on the solid phase with a detectionsystem that detects the bound primary detection agent.

In another aspect of the present invention, there is provided a methodfor performing an indirect rapid enzyme-linked immunosorbent assay(ELISA), comprising coating a solid phase with an antigen dissolved in aquick coating buffer for between about two to twenty minutes, preferablyfor about five minutes; blocking the solid phase with a blocking reagentdissolved in a quick blocking buffer for between about two to tenminutes, preferably for about five minutes; incubating the solid phasewith a primary detection agent in solution and then washing the solidphase to remove any unbound primary detection agent; incubating thesolid phase with a secondary detection agent and then washing the solidphase to remove any unbound secondary detection agent; and detecting thepresence of an agent bound on the solid phase with a detection systemthat detects the bound secondary detection agent.

In a further aspect of the present invention, there is provided a methodfor performing an indirect rapid enzyme-linked immunosorbent assay(ELISA), comprising coating a solid phase with an antigen dissolved in aquick coating buffer for between about two to twenty minutes, preferablyfor about five minutes; blocking the solid phase with a blocking reagentdissolved in a quick blocking buffer for between about two to tenminutes, preferably for about five minutes; incubating the solid phasesimultaneously with a primary detection agent and a secondary detectionagent and then washing the solid phase to remove any unbound primarydetection agent and secondary detection agent; and detecting thepresence of an agent bound on the solid phase with a detection systemthat detects the bound secondary detection agent.

In another aspect of the present invention, there is provided a methodfor performing an indirect rapid enzyme-linked immunosorbent assay(ELISA), comprising coating a solid phase with an antigen dissolved in aquick coating buffer for between about two to twenty minutes, preferablyfor about five minutes; blocking the solid phase with a blocking reagentdissolved in a quick blocking buffer for between about two to tenminutes, preferably for about five minutes; incubating the solid phasewith a primary detection agent in solution and then washing the solidphase to remove any unbound primary detection agent; incubating thesolid phase with a secondary detection agent and then washing the solidphase to remove any unbound secondary detection agent; incubating thesolid phase with a tertiary detection agent and then washing the solidphase to remove any unbound tertiary detection agent; and detecting thepresence of an agent bound on the solid phase with a detection systemthat detects the bound tertiary detection agent.

In still another aspect of the present invention, there is provided amethod for performing an indirect rapid enzyme-linked immunosorbentassay (ELISA), comprising coating a solid phase with an antigendissolved in a quick coating buffer for between about two to twentyminutes, preferably for about five minutes; blocking the solid phasewith a blocking reagent dissolved in a quick blocking buffer for betweenabout two to ten minutes, preferably for about five minutes; incubatingthe solid phase simultaneously with a primary detection agent, asecondary detection agent and a tertiary detection agent and thenwashing the solid phase to remove any unbound primary, secondary andtertiary detection agent; and detecting the presence of an agent boundon the solid phase with a detection system that detects the boundtertiary detection agent.

In still another aspect of the present invention, there is provided amethod for performing a rapid cell-based enzyme-linked immunosorbentassay (C-ELISA), comprising lysing cells in a quick lysis and coatingbuffer; coating a solid phase with cellular proteins released in situ inthe quick lysis and coating buffer for about two to twenty minutes,preferably for about five to ten minutes; blocking the solid phase witha blocking reagent dissolved in a quick blocking buffer for betweenabout two to ten minutes, preferably for about five minutes; incubatingthe solid phase with a primary detection agent in solution; washing thesolid phase to remove any unbound primary detection agent; and detectingthe presence of an agent bound on the solid phase with a detectionsystem that detects the bound primary detection agent.

In still another aspect of the present invention, there is provided amethod for performing a rapid cell-based enzyme-linked immunosorbentassay (C-ELISA), comprising lysing cells in a quick lysis and coatingbuffer; coating a solid phase with cellular proteins released in situ inthe quick lysis and coating buffer for about two to twenty minutes,preferably for about five to ten minutes; blocking the solid phase witha blocking reagent dissolved in a quick blocking buffer for betweenabout two to ten minutes, preferably for about five minutes; incubatingthe solid phase with a primary detection agent in solution and thenwashing the solid phase to remove any unbound primary detection agent;incubating the solid phase with a secondary detection agent and thenwashing the solid phase to remove any unbound secondary detection agent;and detecting the presence of an agent bound on the solid phase with adetection system that detects the bound secondary detection agent.

In still another aspect of the present invention, there is provided amethod for performing a rapid cell-based enzyme-linked immunosorbentassay (C-ELISA), comprising lysing cells in a quick lysis and coatingbuffer; coating a solid phase with cellular proteins released in situ inthe quick lysis and coating buffer for about two to twenty minutes,preferably for about five to ten minutes; blocking the solid phase witha blocking reagent dissolved in a quick blocking buffer for betweenabout two to ten minutes, preferably for about five minutes; incubatingthe solid phase simultaneously with a primary detection agent and asecondary detection agent; washing the solid phase to remove any unboundprimary and secondary detection agent; and detecting the presence of anagent bound on the solid phase with a detection system that detects thebound secondary detection agent.

In another aspect of the present invention, there is provided a methodfor performing an indirect rapid cell-based enzyme-linked immunosorbentassay (C-ELISA), comprising lysing cells in a quick lysis and coatingbuffer; coating a solid phase with cellular proteins released in situ inthe quick lysis and coating buffer for two to twenty minutes, preferablyfor about five minutes; blocking the solid phase with a blocking reagentdissolved in a quick blocking buffer for between about two to tenminutes, preferably for about five minutes; incubating the solid phasewith a primary detection agent in solution and then washing the solidphase to remove any unbound primary detection agent; incubating thesolid phase with a secondary detection agent and then washing the solidphase to remove any unbound secondary detection agent; incubating thesolid phase with a tertiary detection agent and then washing the solidphase to remove any unbound tertiary detection agent; and detecting thepresence of an agent bound on the solid phase with a detection systemthat detects the bound tertiary detection agent.

In still a further aspect of the present invention, there is provided amethod for performing a rapid cell-based enzyme-linked immunosorbentassay (C-ELISA), comprising lysing cells in a quick lysis and coatingbuffer; coating a solid phase with cellular proteins released in situ inthe quick lysis and coating buffer for about two to twenty minutes,preferably for about five to ten minutes; blocking the solid phase witha blocking reagent dissolved in a quick blocking buffer for betweenabout two to ten minutes, preferably for about five minutes; incubatingthe solid phase simultaneously with a primary detection agent, asecondary detection agent and a tertiary detection agent; washing thesolid phase to remove any unbound primary, secondary and tertiarydetection agent; and detecting the presence of an agent bound on thesolid phase with a detection system that detects the bound tertiarydetection agent.

In still another aspect of the present invention, there is provided arapid enzyme-linked immunosorbent assay (ELISA), comprising a quickcoating buffer and a blocking reagent in a quick blocking buffer. Thequick coating buffer is comprised of water and a metal hydroxide suchas, without limitation, sodium hydroxide, potassium hydroxide orrubidium hydroxide. Preferably, the metal hydroxide is sodium hydroxide.The concentration of the metal hydroxide can range from between about0.004 g/l to 40 g/l of buffer, and preferably is about 4 g/l of buffer.The pH of the quick coating buffer can range from between about 10.0 to14.0, and preferably is about 13.0. The quick blocking buffer iscomprised of water and a metal hydroxide selected from the groupconsisting of sodium hydroxide, potassium hydroxide and rubidiumhydroxide, and preferably is potassium hydroxide. The concentration ofthe metal hydroxide can range from between about 0.0056 g/l to 56 g/l ofbuffer, and preferably is about 5.6 g/l of buffer. The pH of the quickblocking buffer can range from between about 10.0 to 14.0, andpreferably is about 13.0.

In still a further aspect of the present invention, there is provided akit for performing a rapid enzyme-linked immunosorbent assay (ELISA), awestern blot assay or a dot blot assay, comprising a quick coatingbuffer and a blocking reagent dissolved in a quick blocking buffer. Theblocking reagent can include, without limitation, non-fat milk, casein,bovine serum albumin, fish gelatin or other suitable chemical reagentsknown in the art. The quick coating buffer is comprised of water and ametal hydroxide such as, without limitation, sodium hydroxide, potassiumhydroxide or rubidium hydroxide. Preferably, the metal hydroxide issodium hydroxide. The concentration of the metal hydroxide can rangefrom between about 0.004 g/l to 40 g/l of buffer, and preferably isabout 4 g/l of buffer. The pH of the quick coating buffer can range frombetween about 10.0 to 14.0, and preferably is about 13.0. The quickblocking buffer is comprised of water and a metal hydroxide selectedfrom the group consisting of sodium hydroxide, potassium hydroxide andrubidium hydroxide, and preferably is potassium hydroxide. Theconcentration of the metal hydroxide can range from between about 0.0056g/l to 56 g/l of buffer, and preferably is about 5.6 g/l of buffer. ThepH of the quick blocking buffer can range from between about 10.0 to14.0, and preferably is about 13.0.

An object of the present invention, therefore, is to provide improvedrapid direct and indirect ELISA methods for the detection andquantification of antibodies by using an improved coating buffer.Accordingly, the coating time can be reduced from more than two hours toabout five minutes.

Another object of the present invention is to provide improved rapiddirect and indirect ELISA methods for the detection and quantificationof antibodies by using an improved blocking reagent. Accordingly, theblocking time can be reduced from one hour to about five minutes.

Still another object of the invention is to provide a novel and rapidcell-based ELISA (C-ELISA) method for the detection and quantificationof proteins released in situ from cells by using a novel cell lysis andcoating reagent, in which the cells are lyzed, the cellular proteins arereleased and coated onto the surface of plate wells at the same time ina single buffer in as little as five minutes. This removes both the cellfixing step, cell permealizing step and the wash steps related to thosetwo steps.

It is a further object of the invention to provide an improved rapiddirect and indirect ELISA method for the detection and quantification ofantibodies by using both the improved coating buffer and the blockingreagents. Accordingly, the time can be reduced from more than four hoursto about two hours or less (e.g., to about one hour).

A further object of the present invention is to provide an improvedrapid indirect ELISA method for the detection and quantification ofantibodies by integrating conventional indirect ELISA containing twosteps into a one step method, and thus providing a more efficientprocess.

BRIEF DESCRIPTION OF THE DRAWINGS

A full understanding of the invention can be gained from the followingdescription of the preferred embodiments when read in conjunction withthe accompanying drawings in which:

FIG. 1 schematically illustrates procedures of a classical direct ELISA;

FIG. 2 schematically illustrates procedures of a direct ELISA process ofthe invention;

FIG. 3 schematically illustrates procedures of a classical indirectELISA;

FIG. 4 schematically illustrates procedures of an indirect ELISA processof the invention;

FIG. 5 schematically illustrates procedures of a further indirect ELISAprocess of the invention; and

FIG. 6 shows an example of an indirect ELISA of the invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art to which this invention pertains. All publications andpatents referred to herein are incorporated by reference.

The invention provides methods and compositions useful for performingantigen or antibody detection or diagnostics using ELISA. In one aspect,the invention provides a significant improvement over conventional ELISAtechniques. The invention provides a method whereby several steps in aclassical ELISA procedure can be performed or completed in a few minutesinstead of hours. The invention also provides a method whereby severalsteps in a classical ELISA procedure time can be combined into one stepor two steps. The methods of the invention greatly reduce the time fordetection assays as well as associated costs.

As used herein “antigen” and “antibody” are to be taken in theirbroadest context. An “antigen” can be any molecule, cell, virus, orparticle. For example, an antigen includes, but is not limited to, achemical molecule, a peptide molecule, a protein molecule, an RNAmolecule, a DNA molecule, a traditional antibody, e.g., two heavy chainsand two light chains, a recombinant antibody or fragment, a bacterialcell, a virus particle, a cell, a particle, and a product comprisingcrosslinking any two or more of the above.

An antigen can be in a pure form, or it can exist in a mixture. Anantigen can be in a modified form (e.g., modified by chemicals) or canbe in an unmodified form.

Reference herein to an “antibody” is to be taken in its broadestcontext. “An antibody” is a polypeptide that binds to “an antigen.” Anantibody includes, but is not limited to, a traditional antibody, afragment of a traditional antibody containing an antigen binding site, arecombinant antibody containing an antigen binding site, a protein whichbinds to an antigen, and a product comprising crosslinking any two ormore of the above.

An antibody can be in a pure form, or it can exist in a mixture. Anantibody can be in a modified form (e.g., modified by a chemical) or canbe in an unmodified form.

The term “detection agent” refers to an agent that is used to detect anantigen or antibody. A detection agent can be either an “antigen” or an“antibody.” A detection agent can be either a labeled “antigen” or“antibody” or an unlabeled “antigen” or “antibody.” Suitable labelingmethods that can be used in the present invention include, withoutlimitation, isotope labeling, chemical modification, enzyme conjugation,fluorescent dye labeling, luminescence and other labeling methodscommonly known by those skilled in the art. Therefore, a detection agentincludes, but is not limited to, a chemical molecule, a peptidemolecule, a protein molecule, an RNA molecule, a DNA molecule, atraditional antibody, a fragment of a traditional antibody containing anantigen binding site, a recombinant antibody containing an antigenbinding site, a protein which binds to an antigen, a bacterial cell, aviral particle, a cell, a particle, and a product comprisingcrosslinking any two or more of the above.

A detection agent can be in a pure form, or it can be an impure form(e.g., contained in a mixture with other compounds or materials). Adetection agent can be in a modified form or can be an unmodified form.According to the order of a “detection agent” used in a method, a“detection agent” can be referred as “a primary detection agent,”“secondary detection agent,” “a tertiary detection agent” or “a fourthdetection agent,” and the like.

The term “detection system” refers to a system which can be used to givea readout comprising information related to the quantity or quality of aprotein or agent in a sample (e.g., a blot, cell and the like). Thechoice of a detection system depends on the choice of the detectionagent used in a method of the invention. For example, a detection systemincludes, but is not limited to, X-ray film or other beta/gammasensitive material if the detection agent is isotope-labeled; if thedetection agent is enzyme-labeled, a chemical reaction which can resultin color or a chemiluminescence signal that can be detected by, forexample, a CCD camera, visual inspection or other device capable ofsensing a signal can be used; and if the detection agent isfluorescence-labeled, a fluorescence microscope, a fluorescence cellsorter, a fluorescence scanner or camera can be used.

The invention provides compositions useful in ELISAs. One of thecompositions is referred to herein as a quick coating buffer. A quickcoating buffer of the invention comprises sodium hydroxide (NaOH) andwater. Sodium hydroxide may be substituted with similar elements knownin the art that function in solution in substantially the same way. Forexample, sodium hydroxide can be substituted with potassium hydroxide(KOH) or rubidium hydroxide (RbOH). In one aspect, the quick coatingbuffer comprises about 0.004 grams to 40 grams per liter of buffer,typically about 4 grams. The quick coating buffer can have a pH in therange of about 10.0 to about 14.0, but typically is about 13.0.

A quick coating buffer of the invention can be bottled and used astypically performed in research and diagnostic laboratories. The quickcoating buffer of the present invention is made in sterile water ordistilled water that is sterile filtered and/or autoclaved. The quickcoating buffer of the invention can be used with ELISA assays. The quickcoating buffer can be included in an article of manufacture or kit foruse in ELISAs and the like.

The invention provides a second composition useful in ELISAs. Thecomposition is referred to herein as a quick blocking buffer. A quickblocking buffer of the invention comprises potassium hydroxide (KOH) andwater. Potassium hydroxide may be substituted with similar elementsknown in the art that function in solution in substantially the sameway. For example, potassium hydroxide can be substituted with sodiumhydroxide (NaOH) or rubidium hydroxide (RbOH). In one aspect, the quickblocking buffer comprises about 0.0056 grams to 56 grams per liter ofbuffer, typically about 5.6 grams. The quick blocking buffer can have apH in the range of about 10.0 to about 14.0, but typically is about13.0.

The invention provides a third composition useful in Cell-based ELISA(C-ELISA). The composition is referred to herein as a quick lysis andcoating buffer. A quick lysis and coating buffer of the inventioncomprises potassium hydroxide and water. Potassium hydroxide may besubstituted with similar elements known in the art that function insolution in substantially the same way. For example, potassium hydroxidecan be substituted with sodium hydroxide (NaOH) or rubidium hydroxide(RbOH). In one aspect, the quick lysis and coating buffer comprisesabout 0.0056 grams to 56 grams per liter of buffer, typically about 5.6grams. The quick lysis and coating buffer can have a pH in the range ofabout 10.0 to about 14.0, but typically is about 13.0.

The quick coating buffer, quick blocking buffer a quick lysis andcoating buffer of the present invention can be the same or different.

A quick blocking buffer of the invention can be bottled and used astypically performed in research and diagnostic laboratories. The quickblocking buffer is made in sterile water or distilled water that issterile filtered and/or autoclaved. The quick blocking buffer of theinvention can be used with ELISA assays. The quick blocking buffer canbe included in an article of manufacture or kit for use in ELISAs andthe like. The quick lysis and coating buffer can also be included in anarticle of manufacture or kit for use in ELISAs and the like.

The invention mainly can be used in three types of ELISA methods: (1)direct ELISA (antibody capture); (2) indirect ELISA; and (3) Cell-basedELISA.

Direct ELISA (antibody capture assay) is one ELISA method. For detectingor quantitating an antigen or a detection agent (e.g. an antibody) thatrecognizes an antigen, the antigen is coated on the wells of microtiterplates and incubated with test solutions containing specific detectionagents. Usually a reporter-molecule labeled primary detection agent isadded to the test solutions containing specific detection agents. Afterincubation, any unbound labeled primary detection agent is washed away.An incubation with a substrate of reporter enzyme also may be needed. Adetection system such as UV, fluorescence, chemiluminescence or othermethods is used to detect the bound primary detection agent, which isproportional to the amount of the detection agent to be detected in thetest solution.

A conventional direct ELISA is comprised of the steps enumerated inFIG. 1. Three major steps are needed before the final detection step.These steps comprise an antigen coating step, a blocking step andprimary detection agent binding step. Each of these steps is necessaryin conventional direct ELISA to obtain acceptable results. Inconventional direct ELISA, the coating step takes 2 hours at 37° C. or4° C. o/n. The blocking step takes 1 hour to complete.

The invention provides a direct ELISA that differs from conventionaltechniques in that each of the coating step and the blocking step ofconventional indirect ELISA can be done in just five minutes instead ofone or two hours. The new method can greatly cut down the time requiredfor indirect ELISA analysis.

As shown in FIG. 2, the direct ELISA method of the invention comprises(i) coating a solid phase with an antigen dissolved in the coatingbuffer of the invention (five minutes only are needed); (ii) blocking asolid phase with a blocking reagent dissolved in the blocking buffer ofthe invention (five minutes only are needed); (iii) incubating the solidphase of (ii) with a primary detection agent in a solution followed by(iv) detecting the presence of an agent on the solid phase with adetection system that measures, for example, UV, fluorescence,luminescence, colorimetric or other signal to detect the bound primarydetection agent.

Indirect ELISA (detection agent capture assay) is one ELISA method thatcommonly is used for screening and titer determination of antibodiesduring the course of their production (Douillard, J. Y. and Hoffman, T.,1983). For detecting a detection agent (e.g. an antibody) thatrecognizes an antigen, the antigen is coated on the wells of microtiterplates and incubated with test solutions containing specific detectionagents. Unbound detection agents are washed away. Then a secondincubation with a solution containing a secondary detection agent (e.g.alkaline phosphatase conjugated to protein A, protein G, or antibodiesagainst the detection agents of interest) is needed. After incubation,unbound labeled secondary detection agent is washed away. An incubationwith a substrate of reporter enzyme also may be needed. A detectionsystem such as UV, fluorescence, chemiluminescence or other methods isused to detect the bound secondary detection agent, which isproportional to the amount of the detection agent to be detected in thetest solution.

A conventional indirect ELISA is comprised of the steps enumerated inFIG. 3. In conventional indirect ELISA, four major steps are neededbefore the final detection step. These steps comprise an antigen coatingstep, a blocking step, primary detection agent binding step andsecondary detection agent binding step. Each of these steps is necessaryin conventional indirect ELISA to obtain acceptable results. Theblocking step blocks remaining hydrophobic binding sites on the solidphase to prevent non-specific protein binding of the detection agentused for detection of the target protein, thereby reducing backgroundand/or preventing false positive results. The primary detection agentand secondary detection agent are incubated with the solid phaseseparately, and then washed away to avoid non-specific binding and toreduce the background.

The invention provides an indirect ELISA that differs from conventionaltechniques in that each of the coating step and the blocking step ofconventional indirect ELISA can be done in just five minutes instead ofone or two hours. The new method can greatly cut down the time requiredfor indirect ELISA analysis.

As shown in FIG. 4, the indirect ELISA method of the invention comprises(i) coating a solid phase with an antigen dissolved in the coatingbuffer of the invention (five minutes only are needed); (ii) blocking asolid phase with a blocking reagent dissolved in the blocking buffer ofthe invention (five minutes only are needed); (iii) incubating the solidphase of (ii) with a primary detection agent in a solution followed by(iv) incubating the solid phase of (iii) with a secondary detectionagent; and (iv) detecting the presence of an agent on the solid phasewith a detection system that measures, for example, UV, fluorescence,luminescence, colorimetric or other signal to detect the bound secondarydetection agent.

In another embodiment of the invention, as shown in FIG. 5, the indirectELISA of the invention comprises (i) coating a solid phase with anantigen dissolved in the coating buffer of the invention (five minutesonly are needed); (ii) blocking a solid phase with a blocking reagentdissolved in the blocking buffer of the invention (five minutes only areneeded); (iii) incubating the solid phase of (ii) with a primarydetection agent and a secondary detection agent; and (iv) detecting thepresence of an agent on the solid phase with a detection system thatmeasures, for example, UV, fluorescence, luminescence, colorimetric orother signal to detect the bound secondary detection agent.

In yet another embodiment of the invention, the indirect ELISA method ofthe invention comprises (i) coating a solid phase with an antigendissolved in the coating buffer of the invention (five minutes only areneeded); (ii) blocking a solid phase with a blocking reagent dissolvedin the blocking buffer of the invention (five minutes only are needed);(iii) incubating the solid phase of (ii) with a primary, secondary, andtertiary detection agent in a solution; and (iv) detecting the presenceof an agent on the solid phase with a detection system that measures,for example, UV, fluorescence, luminescence, colorimetric or othersignal to detect the bound secondary or tertiary detection agent.

A conventional Cell-based ELISA (C-ELISA) is comprised of five majorsteps before the final detection step. These steps comprise a cellfixing step, a cell permealizing step, a blocking step, primarydetection agent binding step and secondary detection agent binding step.Each of these steps is necessary in conventional Cell-based ELISA(C-ELISA) to obtain acceptable results. The cell fixing step will holdcells to the bottom of plate wells so that they will not be washed awayin the process. The cell permealizing step will make the cellularproteins available for immunoassays. The blocking step blocks remaininghydrophobic binding sites on the solid phase to prevent non-specificprotein binding of the detection agent used for detection of the targetprotein, thereby reducing background and/or preventing false positiveresults. The primary detection agent and secondary detection agent areincubated with the solid phase separately, and then washed away to avoidnon-specific binding and to reduce the background.

The invention provides a Cell-based ELISA (C-ELISA) that differs fromconventional techniques in that a cell fixing step and a cellpermealizing step of conventional Cell-based ELISA are replaced by asingle lysis and coating in just five to ten minutes. The blocking stepof conventional Cell-based ELISA also can be done in just five minutesinstead of one or two hours. The improved method of the presentinvention can greatly cut down the time required for Cell-based ELISA(C-ELISA).

The Cell-based ELISA (C-ELISA) method of the invention comprises (i)lysing cells and coating a solid surface with the cellular proteinsreleased in situ in the lysis and coating buffer of the invention (fiveto ten minutes only are needed); (ii) blocking a solid phase with ablocking reagent dissolved in the blocking buffer of the invention (fiveminutes only are needed); (iii) incubating the solid phase of (ii) witha primary detection agent in a solution followed by (iv) incubating thesolid phase of (iii) with a secondary detection agent; and (v) detectingthe presence of an agent on the solid phase with a detection system thatmeasures, for example, UV, fluorescence, luminescence, colorimetric orother signal to detect the bound secondary detection agent.

In another embodiment of the invention, the Cell-based ELISA (C-ELISA)of the invention comprises (i) lysing cells and coating a solid surfacewith the cellular proteins released in situ in the lysis and coatingbuffer of the invention (five to ten minutes only are needed); (ii)blocking a solid phase with a blocking reagent dissolved in the blockingbuffer of the invention (five minutes only are needed); (iii) incubatingthe solid phase of (ii) with a primary detection agent and a secondarydetection agent; and (iv) detecting the presence of an agent on thesolid phase with a detection system that measures, for example, UV,fluorescence, luminescence, colorimetric or other signal to detect thebound secondary detection agent.

In yet another embodiment of the invention, the Cell-based ELISA(C-ELISA) of the invention comprises (i) lysing cells and coating asolid surface with the cellular proteins released in situ in the lysisand coating buffer of the invention (five to ten minutes only areneeded); (ii) blocking a solid phase with a blocking reagent dissolvedin the blocking buffer of the invention (five minutes only are needed);(iii) incubating the solid phase of (ii) with a primary detection agent,a secondary detection agent and a tertiary detection agent in asolution; and (iv) detecting the presence of an agent on the solid phasewith a detection system that measures, for example, UV, fluorescence,luminescence, colorimetric or other signal to detect the bound secondaryor tertiary detection agent.

In the above-mentioned embodiments, those skilled in the art will knowhow to select a detection agent for a specific antigen. A detectionagent can be, but is not limited to, a chemical molecule, a peptidemolecule, a protein molecule, an RNA molecule, a DNA molecule, anantibody, a fragment of an antibody, a recombinant antibody, a bacteriacell, a virus particle, a cell, or a particle. One of the most commonlyused detection agents is an antibody. Antibodies can be derived fromdifferent species, and they include, but are not limited to, rabbit,mouse, rat, sheep, goat, and chicken antibodies. Commercially availableantibodies to a wide variety of antigens are known in the art.

Another one of the more commonly used detection agents comprise proteinA, G, L, A/G or other antibody-binding polypeptides. These polypeptidescan bind to the conserved region in an antibody. Yet another one of themost used detection agents is avidin or strepavidin. Avidin orstreptavidin can bind to biotinylated antibodies or polypeptides. Thedetection agent in the above-mentioned embodiments can be a labeleddetection agent or an unlabeled detection agent.

In the above-mentioned embodiments, those skilled in the art will knowthat there are a variety of labeling methods for a detection agent. Thelabeling methods include, but are not limited to, an enzyme such ashorseradish peroxidase (HRP), alkaline phosphatase (AP),beta-galactosidase or other enzymes. A detection agent also can belabeled with radioactive isotopes of iodine or other isotopes. Adetection agent also can be labeled by a fluorochrome (a fluorescentdye) that can be detected by fluorescence microscope or fluorometer orscanner or camera. A detection agent also can be labeled by a lumichromewhich can be detected by luminescence methods. Alternatively, adetection agent also can be labeled by biotin, which can bind to avidinor streptavidin.

In the above-mentioned embodiments, those skilled in the art will beaware of different detection systems used in ELISAs that can be appliedto the methods of the invention described herein. These detectionsystems include, but are not limited to, detection systems usingchromogenic reactions of reporter enzymes such as horseradish peroxidase(HRP) and alkaline phosphatase (AP). The reporter enzymes can usedifferent substrates for chromogenic detection. For example, HRP can use4 CN (4-chloro-1-napthol), DAB/NiCl2 (3,3′-diaminobenzidine/NiCl2), orTMB as substrates for chromogenic detection.

In the above-mentioned embodiments, those skilled in the art will beaware of modifications to further improve the signal to noise ratio.These modifications include, but are not are limited to, adding one ormultiple steps to the above embodiment.

Examples of blocking agents useful in the invention include, but are notlimited to, non-fat milk, casein, BSA, or fish gelatin, or otherchemical reagents. The pH of the working solution can be in the range of10 to 14, typically 13.0.

The invention also provides kits comprising one or more componentsuseful for performing an ELISA and instructions for carrying out amethod of the invention. For example, such instructions can includemethods for preparing a coating buffer and a blocking buffer of theinvention. In another aspect, the kit may be compartmentalized toreceive a coating buffer and/or a blocking buffer and one or morecomponents for performing an ELISA.

Various embodiments of the invention have now been described. It is tobe noted, however, that this description of these specific embodimentsis merely illustrative of the principles underlying the inventiveconcept. It is therefore contemplated that various modifications of thedisclosed embodiments will, without departing from the spirit and scopeof the invention, be apparent to persons skilled in the art.

The present invention is more particularly described in the followingnon-limiting examples, which are intended to be illustrative only, asnumerous modifications and variations therein will be apparent to thoseskilled in the art.

Example I Indirect ELISA for the Titration of Antibody

Purified GST protein (Genscript, Cat. No. Z02039) was coated on 96-wellplates at 4 μg/ml following standard method (C and D) and the quickcoating method of the invention (A and B), respectively. The plate wasblocked following standard method (C and D) and the quick coating methodof the invention (A and B), respectively. Rabbit anti GST serum wasdiluted and added to the plate wells for 1 hour at 37° C. for classicmethod, and 30 minutes at room temperature for the quick method of theinvention. Plates were washed three times, Goat anti rabbit HRP(GenScript, Cat. A00098) was diluted 1:10000 and added to the platewells for 1 hour at 37° C. for classic method, and 20 minutes at roomtemperature for the quick method of the invention. Finally the platewells were washed and developed with TMB system (Genscript, Cat. No.M00078). The absorbance at 450 nm was measured using a microtiter platespectrophotometer. The results are shown in FIG. 6.

It will be appreciated by those skilled in the art that changes could bemade to the embodiments described above without departing from the broadinventive concept thereof. It is understood, therefore, that thisinvention is not limited to the particular embodiments disclosed, but itis intended to cover modifications that are within the spirit and scopeof the invention, as defined by the appended claims.

1. A method for performing a rapid cell-based enzyme-linkedimmunosorbent assay (C-ELISA), comprising the steps of: (a) lysing cellsin a quick lysis and coating buffer, wherein the quick lysis and coatingbuffer comprises water and a metal hydroxide selected from the groupconsisting of sodium hydroxide, potassium hydroxide and rubidiumhydroxide; (b) coating a solid phase with cellular proteins released insitu in the quick lysis and coating buffer; (c) blocking the solid phasewith a blocking reagent dissolved in a quick blocking buffer, whereinthe quick blocking buffer comprises water and a metal hydroxide selectedfrom the group consisting of potassium hydroxide, sodium hydroxide andrubidium hydroxide; (d) (i) incubating the solid phase with a primarydetection agent in solution; washing the solid phase to remove anyunbound primary detection agent; and detecting the presence of an agentbound on the solid phase with a detection system that detects the boundprimary detection agent; or (ii) incubating the solid phase with aprimary detection agent in solution; washing the solid phase to removeany unbound primary detection agent; incubating the solid phase with asecondary detection agent in solution; washing the solid phase to removeany unbound secondary detection agent; and detecting the presence of anagent bound on the solid phase with a detection system that detects thebound secondary detection agent; or (iii) incubating the solid phasesimultaneously with a primary detection agent and a secondary detectionagent in solution; washing the solid phase to remove any unbound primarydetection agent and secondary detection agent; and detecting thepresence of an agent bound on the solid phase with a detection systemthat detects the bound secondary detection agent; or (iv) incubating thesolid phase with a primary detection agent in solution; washing thesolid phase to remove any unbound primary detection agent; incubatingthe solid phase with a secondary detection agent in solution; washingthe solid phase to remove any unbound secondary detection agent;incubating the solid phase with a tertiary detection agent in solution;washing the solid phase to remove any unbound tertiary detection agent;and detecting the presence of an agent bound on the solid phase with adetection system that detects the bound tertiary detection agent; or (v)incubating the solid phase simultaneously with a primary detectionagent, a secondary detection agent and a tertiary detection agent insolution; washing the solid phase to remove any unbound primarydetection agent, secondary detection agent and tertiary detection agent;and detecting the presence of an agent bound on the solid phase with adetection system that detects the bound tertiary detection agent.
 2. Themethod according to claim 1, wherein the solid phase is coated with thecellular proteins in the quick lysis and coating buffer for betweenabout five minutes to ten minutes and the solid phase is blocked withthe blocking reagent in the quick blocking buffer for about fiveminutes.
 3. The method according to claim 1, wherein the metal hydroxidehas a concentration ranging from between about 0.0056 g/l to 56 g/l ofquick lysis and coating buffer, and wherein the quick lysis and coatingbuffer has a pH ranging from between about 10.0 to 14.0.
 4. The methodaccording to claim 3, wherein the metal hydroxide is potassiumhydroxide.
 5. The method according to claim 4, wherein the potassiumhydroxide concentration is about 5.6 g/l of buffer, and wherein thequick lysis and coating buffer has a pH of about 13.0.
 6. The methodaccording to claim 1, wherein the solid phase is coated with thecellular proteins in the quick lysis and coating buffer for betweenabout two minutes to twenty minutes and the solid phase is blocked withthe blocking reagent in the quick blocking buffer for about two totwenty minutes.